Platinating and alkylating agents are standard first-line chemotherapy for many forms of human cancer. Typically, patients initially respond well to these agents; however, many can develop resistance and experience relapse, which requires a change in drug regimen to combat the relapsed cancer. In addition, the high doses of these drugs required for their anti-cancer effects can result in toxic side effects in other tissues throughout the body, limiting both the short- and long-term effectiveness of first-line agents. Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication without repairing the damage. In the context of cancer treatment, TLS promotes survival of tumor cells by allowing replication over platinum and alkyl DNA adducts, which results in increased mutations in surviving tumor cells and acquisition of resistance to the first-line agent. Disruption of TLS sensitizes cancers to genotoxic agents and reduces mutagenesis in tumors, suggesting that combination therapy with an inhibitor of TLS could enhance the efficacy of first-line agents and prevent chemoresistance. As such, small molecule inhibitors of TLS are emerging as a new class of adjuvant agents for first-line cancer chemotherapy. Assembly of the multi-protein complex that mediates TLS is controlled by the DNA polymerase Rev1, which serves as the central scaffold to maintain the complex through multiple protein-protein interactions (PPIs). Several essential steps of TLS are mediated through PPIs between the C-terminal domain of Rev1 (Rev1-CT) and Rev-1 interacting regions (RIR) from multiple TLS DNA polymerases. Previous studies have demonstrated that suppression of Rev1 expression in vitro and in vivo sensitizes cancer cells to genotoxic chemotherapeutics and decreases acquired drug resistance in tumors. Recent collaborative research in our labs has led us to identify the first reported small molecules that inhibit TLS by disrupting the Rev1-CT/RIR PPI through direct binding to Rev1-CT at the RIR interface. These compounds increase cisplatin sensitivity and reduce cisplatin-induced mutagenesis in human cancer cells. Our results demonstrate that the Rev1-CT/RIR interface is a druggable PPI and they validate that its disruption enhances the anti-cancer effects of first-line genotoxic agents. Within this context, the overall goal of our studies is to apply a comprehensive interdisciplinary approach to develop our lead TLS inhibitors as a new class of anti-cancer therapeutics. In pursuit of this goal, we will undertake the following specific aims: (1) synthesize and characterize improved small molecule inhibitors of the Rev1-CT/RIR PPI, (2) evaluate Rev1-CT/RIR inhibitors using in vitro biochemical and structural approaches, (3) probe cellular anti-TLS and anti-cancer activities of the Rev1-CT/RIR inhibitors, and (4) perform in vivo studies on optimal compounds. We anticipate that these studies will identify small molecules with enhanced anti-TLS activity and improved drug-like properties as promising cancer chemotherapeutics.